Andrea Natali

Context: Type 2 diabetes (T2D) markedly increases the risk of cardiovascular disease (CVD). Circulating microRNAs (miRNAs) are promising biomarkers for patient stratification and may provide mechanistic insight into dysregulated pathways in T2D-associated CVD. Objective: This work aimed to identify circulating miRNA signatures in T2D patients with CVD (T2D-CVD) vs T2D patients without CVD using a novel network-based medicine approach. Methods: Patients (n = 110) were stratified into T2D and T2D-CVD groups based on the absence or presence of macrovascular complications. Circulating miRNAs, proinflammatory cytokines, and growth factors were profiled in a discovery cohort (n = 80) and confirmed in a validation cohort (n = 30). Differential expression, network-based approaches, and pathway enrichment analyses identified miRNA signatures, coexpression communities, hub miRNAs, and associated signaling pathways. Results: T2D-CVD patients showed a set of elevated inflammatory cytokines and circulating miRNAs. Particularly, 19 miRNAs were differentially expressed between T2D-CVD and T2D patients. Pathway enrichment of these miRNAs highlighted Hippo signaling together with transforming growth factor-β, mechanistic target of rapamycin, Wnt, and PI3K-Akt pathways. Network analysis identified a coexpression community of 34 miRNAs, including hub miRNAs let-7a-5p, miR-132-3p, and miR-1306-3p, all linked to Hippo signaling. Key miRNA interactions were validated, and differentially expressed and hub miRNAs both correlated with inflammatory mediators such as PAR-1, CXCL1, and NEMO, which are linked to Hippo pathway regulation. Conclusion: This study identified a network of circulating miRNAs associated with inflammatory cytokines and Hippo signaling pathway in T2D-CVD. These findings reveal novel molecular aspects of the systemic inflammation characterizing T2D-CVD and support the potential of Hippo-related circulating miRNAs as mechanistic biomarkers for cardiovascular risk in T2D.

Aims: Inflammasome activation has emerged as a promising therapeutic target to reduce residual cardiovascular (CV) risk in patients on guideline lipid-lowering therapy. IL-1β and IL-6 have been intensively studied, whereas the role of the inflammasome-related cytokine IL-18 has gained less attention. Here we aimed to assess the associations of IL-18 with risk of coronary events and severity of atherosclerosis. Methods and results: The study included 4742 participants participating in a prospective Swedish Malmö Diet and Cancer (MDC) cohort study, 54 219 general population participants in the UK Biobank, and 1500 participants with Type 2 diabetes and/or known CV disease in the European multicentre SUMMIT VIP imaging study. Plasma levels of IL-18 were analysed using proximity extension assay and atherosclerosis by carotid ultrasound. The incidence of MI and CV death was registered during a 23-year follow-up for the MDC cohort and for a 15-year follow-up for the UK Biobank cohort. There were 617 cases of MI and 644 CV deaths in the MDC cohort and 2454 cases of MI and 1537 CV deaths in the UK Biobank cohort. Elevated IL-18 was associated with an increased risk of MI and CV death independent of hsCRP and IL-6 in both cohorts. In Cox proportional hazards regression models, this association was independent of major CV risk factors in the UK Biobank but not in the MDC cohort. IL-18 did not provide clinically meaningful risk reclassification in the UK Biobank cohort. The association of IL-18 with carotid atherosclerosis in the SUMMIT VIP cohort was weaker than that of IL-6. Conclusion: IL-18 is independently associated with risk of MI and CV death in the general population but does not improve clinical risk stratification. Targeting IL-18 directly or through inflammasome inhibition represents a promising approach for treatment of residual inflammatory risk in high-risk atherosclerosis patients that merits to be evaluated in future randomized clinical trials.

Aims/hypothesis: Hepatic glucose flux plays a crucial role in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD), promoting de novo lipogenesis, inflammation and fibrosis. This study aimed to evaluate the kinetics of oral glucose absorption and one of its key modulators, gastric emptying, in individuals with early-stage MASLD vs matched control individuals. Methods: We quantified glucose metabolic fluxes during a 75 g OGTT using stable isotopes in individuals with MASLD without fibrosis and in healthy control individuals. In a separate cohort, we measured the gastric emptying rate using the ¹³ C-acetate breath test during an OGTT and estimated hepatic steatosis risk. Results: Compared with the control group, in the MASLD group the rate of appearance of oral ingested glucose (RaO) normalised to body weight was 34% higher at 1 h post-OGTT (+318±142 µmol/kg, p =0.031), resulting in a 52% increase in total glucose absorption (+6.4±1.8 g, p =0.001). Participants with MASLD exhibited reduced glucose clearance relative to plasma insulin levels but preserved post-load suppression of endogenous glucose production, indicating peripheral rather than hepatic insulin resistance. Among glucose metabolic fluxes, RaO showed the strongest association with prevalent MASLD, with each 1-SD increase in 1 h RaO being associated with fivefold higher odds of MASLD (OR 4.99 [95% CI 1.44, 31.57], p =0.036), independent of potential confounders. Gastric emptying rate was not associated with hepatic steatosis risk. Conclusions/interpretation: Oral glucose absorption is augmented in individuals with MASLD without fibrosis, apparently unrelated to accelerated gastric emptying. This metabolic alteration may represent an early driver of MASLD pathogenesis, preceding hepatic insulin resistance. Future research should investigate whether modulation of intestinal glucose absorption confers therapeutic benefits in MASLD.

Aims/hypothesis Sodium–glucose cotransporter 2 (SGLT2) inhibitors provide cardiovascular and renal protection in type 2 diabetes and chronic kidney disease (CKD). Although both excess and restricted sodium intake are linked to adverse outcomes, the interaction of sodium intake with SGLT2 inhibitors has not been explored. This study aimed to examine how dietary sodium intake affects cardiorenal outcomes and whether canagliflozin modifies these effects. Methods A post hoc analysis of the CREDENCE trial (median follow-up 2.6 years) was conducted in individuals with type 2 diabetes and CKD randomised to canagliflozin 100 mg or placebo. Using a validated formula, we estimated daily sodium intake from urine in 2573 participants, divided into low–normal sodium (LNS; n=1286) and high sodium (HS; n=1287) groups. Outcomes included the following: cardiovascular death or hospitalisation for heart failure; heart failure alone; a composite renal outcome; and all-cause death. Cox models were adjusted for confounders. Sodium intake was additionally analysed as a continuous variable to assess non-linearity. Results In the placebo group, LNS intake increased the risk of heart failure/cardiovascular death vs HS (adjusted HR [adjHR] 1.56 [95% CI 1.10, 2.23]). Canagliflozin significantly reduced this risk in the LNS group (adjHR 0.48 [95% CI 0.33, 0.70]) but not in the HS group (adjHR 1.05 [95% CI 0.73, 1.53]). Similar patterns were seen for heart failure alone. Sodium intake had no effect on renal outcomes, while canagliflozin reduced renal risk in both the LNS group and the HS group. Neither sodium intake nor canagliflozin influenced all-cause mortality. Continuous modelling revealed a near-linear rise in heart failure/cardiovascular death risk as sodium intake decreased in placebo recipients, while this gradient was flattened with canagliflozin. Conclusions/interpretation In individuals with type 2 diabetes and CKD, LNS intake increases the risk of heart failure and cardiovascular death, while renal outcomes are unaffected by sodium intake. Canagliflozin mitigates the increased cardiovascular risk in individuals with LNS intake, while offering renal protection irrespective of dietary sodium. Trial registration Clinicaltrial.gov NCT02065791 Graphical Abstract

Aims/hypothesis Fasting hyperinsulinaemia is a key feature of obesity and is implicated in diabetes progression. However, the following aspects of insulin secretion remain unclear: (1) which index of obesity is most important; (2) what is the shape of the dose–response curve between obesity and insulin secretion; (3) what physiological mechanisms sustain insulin hypersecretion; (4) what are the underlying causes; and (5) whether sex-related differences exist. Methods We analysed data from 1250 healthy participants (547 men, 703 women) of the EGIR-RISC cohort followed up for 3.5 years, with age 30–60 years and BMI 18.5–40.0 kg/m². Assessments included body composition, insulin secretion, beta cell function modelling from an OGTT and clamp-derived insulin sensitivity. Endogenous glucose production (EGP) was measured in a subset of 368 participants. Multivariable regression models and stratifications for BMI, body fat per cent, WHR and fat mass were applied to evaluate the effect of obesity on insulin secretion and beta cell function. Results The impact of obesity on fasting insulin secretion (FIS) was continuous across the full spectrum of BMI and WHR values and was greater in men than women. Among adiposity indices, fat mass (standardised β coefficient [Stβ] 0.27, ppppp<0.002 for both) but not in weight gainers (−0.06 ± 1.1 U/h). Peripheral insulin resistance, plasma NEFA and leptin accounted for only part of obesity’s effect on insulin secretion. Subset analysis of fasting and clamp EGP data suggested a rightwards shift in the dose–response curve across fat mass quintiles, indicating progressive hepatic glucose overproduction despite a preserved hepatic insulin response. Conclusions/interpretation The effect of body mass on insulin secretion is continuous, more pronounced in men, driven by fat mass and waist, sustained by hyperglycaemia and by an upregulation of beta cell insulin secretion and is only partially explained by typical hormonal and metabolic consequences of obesity. We suggest that hepatic glucose overproduction contributes to the fasting hyperinsulinaemia observed in individuals with obesity. Graphical Abstract

Purpose Glucagon-like peptide 1 receptor agonists (GLP-1 RAs) are effective drugs for weight loss and management of obesity-related comorbidities. Their role in 24-hour energy metabolism remains unclear. This study evaluated the effect of liraglutide treatment on 24-hour energy metabolism and body composition in a real-life clinical setting. Methods This prospective study enrolled 11 patients with obesity (8 females; mean age 49 ± 9 years; weight 103 ± 18 kg) treated with liraglutide for 6 months at clinically titrated doses at the Obesity and Lipodystrophy Center, University Hospital of Pisa. Measurements of 24-hour energy expenditure (24hEE), 24-hour sleeping metabolic rate (24hSMR), and substrate oxidation (carbohydrates, lipids, proteins) were obtained via whole-room indirect calorimetry prior to start the therapy (V1) and after 6 months (V2). Body composition was assessed by Dual-Energy X-ray Absorptiometry (DXA) at V1 and V2. Results At V2, participants showed significant weight loss (− 10.5 kg, p < 0.001), primarily driven by a decrease in total fat mass (− 8.7 kg, p < 0.001), with a marked reduction in trunk fat mass (− 5.1 kg, p < 0.001). A modest yet statistically significant reduction in total lean soft tissue was also observed (− 1.7 kg, p = 0.02). No changes in 24hEE and 24hSMR could be detected. Fat oxidation increased (+ 352 kcal/d, p = 0.03), while carbohydrate oxidation decreased (− 422 kcal/d, p = 0.003), and protein oxidation remained stable. Conclusion Liraglutide induces significant weight loss in patients with obesity, primarily through fat mass reduction, while largely preserving lean soft tissue. These changes are accompanied by a shift toward fat oxidation, without relevant variations in 24hEE.

Aims: Circadian heart rate (HR) fluctuations are associated with cardiovascular health. We examined their relationship with microvascular disease and long-term survival in patients with diabetes. Methods and results: In this secondary analysis from the CHAMP1ON cohort of 497 adults with metabolic disease, 349 participants who had type 1 or type 2 diabetes, baseline 24-h ambulatory blood pressure and HR monitoring (ABPM), and survival data over a 21-year observational follow-up were included. Clinical features, microvascular complications, and mortality rates were examined in participants with low circadian HR fluctuations [24-h HR standard deviation (SD) below the median of 30.4] and blunted nocturnal HR dip (<10%). Low 24-h HR SD and blunted nocturnal HR dip were associated with an adverse cardiometabolic risk profile and 12–23% higher prevalence of cardiac autonomic neuropathy and nephropathy. After 6251 person-year follow-up [21.0 (14.0–21.0) years], a total of 136 (39%) deaths occurred, of which 100 (68%) of cardiovascular cause. The low 24-h HR SD group had a higher risk for both cardiovascular [adjusted hazard ratio (aHR) 2.00, 95% confidence interval (CI) 1.30–3.08, P = 0.002] and all-cause mortality (aHR 1.61, 95% CI 1.13–2.29, P = 0.009), compared with high 24-h HR SD. Similarly, patients with blunted nocturnal HR dip had a higher risk for cardiovascular (aHR 1.63, 95% CI 1.08–2.46, P = 0.019) and all-cause mortality (aHR 1.69, 95% CI 1.20–2.38, P = 0.003), compared with those with preserved nocturnal HR dip. Conclusion: Impaired circadian HR fluctuations are associated with microvascular disease and long-term cardiovascular and all-cause mortality in diabetes. The ABPM-derived HR measures may provide a widely available and inexpensive risk stratification tool in this high-risk population.